FACS - Doxorubicin interference in PI channel

[pjberlin]

I am using cytotoxic drug Doxorubicin(DOX) to induce apoptosis. I want to Discriminate Live, Early and Late Apoptotic and Dead cells post treatment with DOX and analyze using FACS.

Propidium Iodide Vs Annexin V - Cy5.

I have Doxorubicin interference in Propidium Iodide channel. Cells uptake DOX upon treatment and become fluorescent in PI channel. I can see a shift of DOX treated cells compared to control cells, about one decade higher towards PI+ quadrant without even staining for PI. This is the issue. SoI wanted to use another Nucleic acid stain Is DAPI good


Suggestion Required!!!!!???

What is the Alternative Nucleic acid stain (Other than PI) best suited for FACS to study apoptotic phases

Can you suggest the best Nucleic acid stain and its company name?



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Propidium Iodide: (excitation 535 nm; emission 617 nm)
Doxorubicin: (excitation 488 nm; emission 520 nm)


using Protocol: (BD FACS CANTO 2)

I am using 2 step Annexin V detection (We dont have Annexin V-FITC)

- 2ul Rabbit anti-Annexin V antibody for 10 min;
ADD/WASH 1mL PBS-BSA-EDTA, Centrifuge 4min.
- 3ul Goat Anti-rabbit conjugated to Cy5 for 10 min;
ADD/WASH 1mL PBS-BSA-EDTA, Centrifuge 4min.
- 4ul Propidium Iodide for 10 Min, WASH in PBS-BSA-EDTA
- Resuspend in 400ul PBS-BSA-EDTA.
- Analyze.

Since I wanted to study apoptosis, i didnot want to use Fixation /permeabilization solution.

[bigdmajor]

DAPI works very well with annexin.